Wiskott-Aldrich Syndrome Patient Research Articles

EBV-related lymphoproliferative disease in Wiskott-Aldrich Syndrome patient

CaseWiskott-Aldrich Syndrome (WAS) is a rare X-linked inborn error of immunity caused by mutations in the WAS gene. It is classically characterized by immunodeficiency, eczema, and micro-thrombocytopenia. It has been known since the 1960s that patients with WAS have an increased risk of lymphoproliferative disease though the exact incidence remains unknown in the American population. Limited case reports have discussed EBV-related lymphoproliferative disease in patients with WAS. We present a case of a 9-year-old boy with known WAS complicated by eczematous rash, thrombocytopenia, recurrent ear infections, and monoclonal gammopathy who was found to have submandibular EBV-associated lymphoid hyperplasia with associated lung and retroperitoneal lymphadenopathy. Family had been offered treatment with hematopoietic stem cell transplant but declined multiple times in the past. Earlier in the year, he presented with possible MIS-C with negative SARS-CoV-2 PCR. He presented to our hospital with mastoiditis and lymphadenopathy. Physical examination showed severe eczema on hands and tender right mastoid. Laboratory evaluation showed thrombocytopenia, elevated IgG of 6290, IgA of 744, IgE of 827, low IgM of 41, and 14% response to pneumococcal titers. He was empirically treated with intravenous antibiotics. ENT performed right postauricular incision and drainage and the culture grew Hemophilus influenza. Throughout his hospital stay, his submandibular lymphadenopathy became more prominent despite treatment. Core needle biopsy of right submandibular lymph node was suggestive of EBV-associated lymphoid hyperplasia. EBV PCR and antibodies were both positive. CT chest, abdomen, and pelvis revealed multifocal pulmonary lymphadenopathy and a diffuse, bilateral nodularity as well as retroperitoneal and mesenteric lymphadenopathy. He was given four doses of weekly Rituximab, which successfully decreased EBV viremia below linear detectability. Immunoglobulin replacement therapy (IgRT) was initiated. Bronchoalveolar lavage and lung biopsy were performed and are results are currently pending. DiscussionWe present a case of a 9-year-old boy with known WAS awaiting transplant who was found to have submandibular EBV-associated lymphoid hyperplasia with associated lung and retroperitoneal lymphadenopathy. While lymphoproliferative disease is a known complication of WAS, EBV-related lymphoproliferative disease in WAS patients has only been reported as case reports and remains a rare but known complication of patient with WAS.

Lymphocytes Utilize Somatic Mutations, Epigenetic Silencing, and the Proteasome to Escape Truncated WASP Expression.

Wiskott-Aldrich Syndrome Protein (WASP) deficiency causes Wiskott-Aldrich Syndrome (WAS), a sex-linked disorder characterized by combined immunodeficiency, microthrombocytopenia, and eczema. Like WASP-deficient humans, WASP-deficient mice produce normal numbers of functionally defective T cells. Here, we report a WAS patient with a novel germline frameshifting WAS mutation encoding a truncated form of WASP lacking the C-terminal cofilin homology (C) and the acidic region (A) domains (WASPΔCA). Although stably overexpressed in embryonic kidney cell lines, WASPΔCA was undetectable in circulating patient leukocytes. Deep sequencing, transcript profiling, and protein degradation analyses demonstrated patientlymphocytes employ an array of genetic, epigenetic, and proteasomal strategies to avoid expressing WASPΔCA.

WAS Promoter-Driven Lentiviral Vectors Mimic Closely the Lopsided WASP Expression during Megakaryocytic Differentiation

Transplant of gene-modified autologous hematopoietic progenitors cells has emerged as a new therapeutic approach for Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency with microthrombocytopenia and abnormal lymphoid and myeloid functions. Despite the clinical benefits obtained in ongoing clinical trials, platelet restoration is suboptimal. The incomplete restoration of platelets in these patients can be explained either by a low number of corrected cells or by insufficient or inadequate WASP expression during megakaryocyte differentiation and/or in platelets. We therefore used in vitro models to study the endogenous WASP expression pattern during megakaryocytic differentiation and compared it with the expression profiles achieved by different therapeutic lentiviral vectors (LVs) driving WAS cDNA through different regions of the WAS promoter. Our data showed that all WAS promoter-driven LVs mimic very closely the endogenous WAS expression kinetic during megakaryocytic differentiation. However, LVs harboring the full-length (1.6-kb) WAS-proximal promoter (WW1.6) or a combination of the WAS alternative and proximal promoters (named AW) had the best behavior. Finally, all WAS-driven LVs restored the WAS knockout (WASKO) mice phenotype and functional defects of hematopoietic stem and progenitor cells (HSPCs) from a WAS patient with similar efficiency. In summary, our data back up the use of WW1.6 and AW LVs as physiological gene transfer tools for WAS therapy.

IL-2 in the tumor microenvironment is necessary for Wiskott-Aldrich syndrome protein deficient NK cells to respond to tumors in vivo.

To kill target cells, natural killer (NK) cells organize signaling from activating and inhibitory receptors to form a lytic synapse. Wiskott-Aldrich syndrome (WAS) patients have loss-of-function mutations in the actin regulator WASp and suffer from immunodeficiency with increased risk to develop lymphoreticular malignancies. NK cells from WAS patients fail to form lytic synapses, however, the functional outcome in vivo remains unknown. Here, we show that WASp KO NK cells had decreased capacity to degranulate and produce IFNγ upon NKp46 stimulation and this was associated with reduced capacity to kill MHC class I-deficient hematopoietic grafts. Pre-treatment of WASp KO NK cells with IL-2 ex vivo restored degranulation, IFNγ production, and killing of MHC class I negative hematopoietic grafts. Moreover, WASp KO mice controlled growth of A20 lymphoma cells that naturally produced IL-2. WASp KO NK cells showed increased expression of DNAM-1, LAG-3, and KLRG1, all receptors associated with cellular exhaustion and NK cell memory. NK cells isolated from WAS patient spleen cells showed increased expression of DNAM-1 and had low to negative expression of CD56, a phenotype associated with NK cells exhaustion. Finally, in a cohort of neuroblastoma patients we identified a strong correlation between WASp, IL-2, and patient survival.

Gene Correction of iPSCs from a Wiskott-Aldrich Syndrome Patient Normalizes the Lymphoid Developmental and Functional Defects.

SummaryWiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency disease caused by mutations in the gene encoding the WAS protein (WASp). Here, induced pluripotent stem cells (iPSCs) were derived from a WAS patient (WAS-iPSC) and the endogenous chromosomal WAS locus was targeted with a wtWAS-2A-eGFP transgene using zinc finger nucleases (ZFNs) to generate corrected WAS-iPSC (cWAS-iPSC). WASp and GFP were first expressed in the earliest CD34+CD43+CD45− hematopoietic precursor cells and later in all hematopoietic lineages examined. Whereas differentiation to non-lymphoid lineages was readily obtained from WAS-iPSCs, in vitro T lymphopoiesis from WAS-iPSC was deficient with few CD4+CD8+ double-positive and mature CD3+ T cells obtained. T cell differentiation was restored for cWAS-iPSCs. Similarly, defects in natural killer cell differentiation and function were restored on targeted correction of the WAS locus. These results demonstrate that the defects exhibited by WAS-iPSC-derived lymphoid cells were fully corrected and suggests the potential therapeutic use of gene-corrected WAS-iPSCs.

Successful second bone marrow transplantation in a Wiskott-Aldrich syndrome patient with systemic vasculitis

Successful second bone marrow transplantation in a Wiskott-Aldrich syndrome patient with systemic vasculitis

Next generation sequencing reveals skewing of the T and B cell receptor repertoires in patients with wiskott-Aldrich syndrome.

The Wiskott–Aldrich syndrome (WAS) is due to mutations of the WAS gene encoding for the cytoskeletal WAS protein, leading to abnormal downstream signaling from the T cell and B cell antigen receptors (TCR and BCR). We hypothesized that the impaired signaling through the TCR and BCR in WAS would subsequently lead to aberrations in the immune repertoire of WAS patients. Using next generation sequencing (NGS), the T cell receptor β and B cell immunoglobulin heavy chain (IGH) repertoires of eight patients with WAS and six controls were sequenced. Clonal expansions were identified within memory CD4+ cells as well as in total, naïve and memory CD8+ cells from WAS patients. In the B cell compartment, WAS patient IGH repertoires were also clonally expanded and showed skewed usage of IGHV and IGHJ genes, and increased usage of IGHG constant genes, compared with controls. To our knowledge, this is the first study that demonstrates significant abnormalities of the immune repertoire in WAS patients using NGS.

Management of autoimmune hemolytic anemia in children and adolescents: A single center experience.

To present and discuss the treatment of autoimmune hemolytic anemia (AIHA). The medical records of all patients (n=19) diagnosed in a tertiary hematology center between 1999 and 2010 were retrospectively reviewed. Median age at diagnosis of AIHA was 5 years (range: 4 months-17 years). In all, 13 patients had primary (idiopathic) AIHA, whereas 2 had primary Evans Syndrome (ES), 2 had autoimmune lymphoproliferative syndrome (ALPS)+ES, and 1 had Wiskott-Aldrich syndrome (WAS)+AIHA. Among the 13 primary idiopathic AIHA patients, 9 recovered following a 4-8-week course of prednisolone treatment without relapses, whereas 3 patients required a longer course of prednisolone. One AIHA patient that was very resistant to prednisolone recovered after cyclosporine A was added to the treatment. All patients with primary idiopathic AIHA were in remission for a median of 3 years (range: 4 months-10 years) at the time this manuscript was written. Among the patients with primary ES, 2 had relapses similar to the ALPS patients. Splenectomy was performed in 1 primary ES patient, who at the time this report was written was also in remission. One ALPS patient required the addition of mycophenolate mofetil due to prednisolone resistance. The WAS patient was treatment resistant and died due to septicemia. Primary AIHA in pediatric patients generally has an acute onset and good response to corticosteroids. Primary or secondary ES has a chronic or relapsing course, and treatment may require other immunosuppressive agents in addition to corticosteroids. Complications of splenectomy must not be underestimated in patients with underlying immunodeficiency. AIHA often causes considerable morbidity and mortality in WAS.

Tyrosine Phosphorylation of WASP Promotes Calpain-Mediated Podosome Disassembly In Myeloid Cells.

AbstractAbstract 1498We have previously shown that a point mutation (Ile294Thr) in the Wiskott Aldrich Syndrome Protein (WASP) detected in a Wiskott Aldrich Syndrome (WAS) patient led to enhanced actin polymerising activity of WASP 1 and well as increased instability2. We also showed that macrophages from this patient displayed an increased number of actin based adhesion structures called podosomes. Additionally, podosomes in macrophages from this patient were extremely dynamic with a high rate of turnover. Based on these results we proposed that the active open conformation of WASP promotes actin polymerisation but it also induces podosome disassembly and adhesion turnover. Although the idea that the same open conformation of WASP leads to both assembly and disassembly of podosomes may be counterintuitive at first, it is possible that for termination of podosomes actin polymerisation and integrin recruitment have to be discontinued and the same constituents of growing podosomes such as active WASP may contribute to the disassembly process. We have also shown that the rapid turn-over of podosomes involves cleavage of WASP by the protease calpain, further supporting a role of WASP in both podosome formation and disassembly. However, the specific signalling mechanisms that make active WASP susceptible to cleavage by calpain leading to podosome disassembly remain unknown and need further clarification. Phosphorylation of WASP Y291 (human) or Y293 (mouse) promotes the open conformation of WASP and results in enhanced actin polymerisation. We now report that in myeloid cells, tyrosine phosphorylation of WASP negatively regulates the stability of podosomes, leading to their calpain-dependent disassembly. Additionally, we found that constitutive phosphorylation of WASP results in extensive degradation in a process that involves calpain. Our data also indicate that phosphorylation of WASP sustains the open/active conformation that promotes cleavage of WASP by calpain. Interestingly, phosphorylated WASP can bind to the WASP interacting protein (WIP) and other proteins that form a complex with WASP in podosomes such as Nck, cortactin. Taken together, our data indicate that in myeloid cells, tyrosine phosphorylation sustains the open conformation of WASP and it enhances its susceptibility to calpain-mediated cleavage preventing accumulation of actin filaments and integrin associated proteins in podosomes. This process facilitates podosome disassembly and cell translocation. These new findings support the key role of WASP as a protein that integrates actin polymerisation and cell adhesion required for mobilisation of myeloid cells during the immune response.Reference List1. Ancliff PJ, Blundell MP, Cory GO et al. Two novel activating mutations in the Wiskott-Aldrich syndrome protein result in congenital neutropenia. Blood 2006;108:2182-2189.2. Moulding DA, Blundell MP, Spiller DG et al. Unregulated actin polymerization by WASp causes defects of mitosis and cytokinesis in X-linked neutropenia. J Exp. Med 2007;204:2213–2224. Disclosures:No relevant conflicts of interest to declare.

Somatic mosaicism in the Wiskott–Aldrich syndrome: Molecular and functional characterization of genotypic revertants

The reasons underlying the occurrence of multiple revertant genotypes in Wiskott-Aldrich syndrome (WAS) patients remain unclear. We have identified more than 30 revertant genotypes in a C995T WAS patient having 10-15% revertant, WAS protein (WASp)-expressing circulating lymphocytes. Of 497 allospecific T-cell clones generated from the peripheral blood, 47.1% carried a revertant sequence. All revertant T-cell clones exhibited restoration of WASp expression. However, anti-CD3-induced proliferative responses varied greatly amongst revertants. Several revertant T-cell clones expressed an internally deleted WASp mutant lacking much of the proline-rich region. This potentially accounts for the reduced anti-CD3 proliferative responses of these T-cell clones. We found no evidence for an increased DNA mutation rate in this patient. We conclude that the diversity of revertant genotypes in our patient does not result from an extraordinary mutation rate and that the amino acid sequence space explored by WASp in revertant T-cells is significantly smaller than might have been predicted from the diversity of revertant genotypes.

WIP Is a Chaperone for Wiskott-Aldrich Syndrome Protein.

WASP, the product of the gene mutated in Wiskott-Aldrich syndrome (WAS), plays a critical role in T cell activation and actin reorganization. WIP is a WASP-interacting protein that binds to Ena-VASP homology 1 (EVH1) domain of WASP. We previously reported functional roles of WIP in the recruitment and activation of WASP in T cells. To investigate a role of WIP in WASP degradation and understand the reason why most missense mutations in WAS patients are in EVH1 domain of WASP, we examined WASP levels in WIP knockout (WIP−/−) T cells and mechanisms of WASP degradation following T cell receptor (TCR) ligation. WASP protein levels, but not mRNA levels, were severely diminished in T cells from WIP−/− mice and were restored by introduction of full-length or WASP binding domain of WIP. A fraction of WASP was cleaved by Ca-dependent protease calpain, ubiquitinated and degraded by the proteasome following TCR ligation. Inducible expression of WASP binding domain of WIP increased WASP levels in vivo and recombinant WIP protein protected WASP from degradation by calpain in vitro. Treatment with the proteasome inhibitors MG132 and bortezomib increased WASP protein levels in T cells from WIP−/− mice and in T and B lymphocytes from two WAS patients with missense mutations (R86H and T45M) that disrupted WIP binding. The calpain inhibitor calpeptin also increased WASP protein levels in activated T cells and B cells from the WAS patients. Despite its ability to increase WASP levels, proteasome inhibitors failed to correct the impaired IL-2 gene expression and low F-actin content in T cells from the R86H WAS patient (published in PNAS , 104, p926–931, 2007). These results demonstrate that WIP stabilizes WASP in normal T cells and that WIP deficiency or impaired WASP-WIP interaction in WAS patients with missense mutation in WIP binding site results in WASP degradation.

Unprecedented diversity of genotypic revertants in lymphocytes of a patient with Wiskott-Aldrich syndrome

AbstractSpontaneous somatic reversions of inherited mutations are poorly understood phenomena that are thought to occur uncommonly in a variety of genetic disorders. When molecularly characterized, revertant cells have rarely exhibited more than one revertant genotype per patient. We analyzed individual allospecific T-cell clones derived from a Wiskott-Aldrich syndrome (WAS) patient identified by flow cytometry to have 10% to 15% revertant, WAS protein–expressing lymphocytes in his blood. Genotypic analysis of the clones revealed a remarkable diversity of deletions and base substitutions resulting in at least 34 different revertant genotypes that restored expression of WASp. A large fraction of these revertant genotypes were also identified in primary T cells purified from peripheral blood. These data suggest that the use of sensitive methods may reveal the presence of wide arrays of individual genotypic revertants in WAS patients and offer opportunities for further understanding of their occurrence.

Two novel mutations identified in the Wiskott-Aldrich syndrome protein gene cause Wiskott-Aldrich syndrome and thrombocytopenia

Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) are rare X-linked genetic disorders caused by mutations of the Wiskott-Aldrich syndrome protein (WASP) gene. Both disorders are clinically characterized by chronic thrombocytopenia of small platelets. WAS is a more severe form of the disorder and also courses with eczema, and immune dysfunction. In the present study, we investigated two novel mutations of the WASP gene in two Spanish families with patients clinically diagnosed as having XLT and WAS, respectively. In one of the families a missense mutation in exon 12 (1488A>G), resulting in the highly conserved glutamic residue changing to glycine at position 485 (D485G), was identified in several members. Notably, a female of this family, with clinical signs of XLT, was determined as the carrier of the mutation and showed a skewed pattern of X-inactivation, preferentially inactivating the X-chromosome carrying the wild-type allele. In the case of the second family, we describe a WAS patient with a single base deletion in exon 2 (266-267delA), resulting in a frameshift (at codon 78) that creates a stop codon at amino acid 127. As a consequence, there was no WASP expression.

Large granular lymphocyte proliferation and revertant mosaicism: two rare events in a Wiskott-Aldrich syndrome patient

We report on a 6 year old patient with an unusual clinical presentation of WAS and oligoclonal proliferation of TCR+ large granular lymphocytes (LGL). Flow cytometry demonstrated two distinct populations of lymphocytes with strongly decreased (WASP-) or normal expression levels of WASP (WASP+), respectively. Molecular analysis confirmed a splice site mutation in intron 2 of the WASP gene in the WASP- cells but not in WASP+ cells. LGL cells were WASP+, suggesting that two independent rare events, somatic revertant mosaicism and LGL expansion, have occurred in a child with WAS. Our report points to diagnostic difficulties in the presence of partial WASP reversions and LGL.

Evidence for Efficacy and Safety of Lentiviral Mediated Gene Transfer in T Cells and CD34+ Cells from Wiskott-Aldrich Syndrome Patients.

Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by eczema, recurrent infections, severe hemorrhages and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Therapy based on the transplant of genetically correct autologous stem cells could represent a valid alternative approach. We investigated the efficacy and the safety of WAS gene transfer using HIV-based lentiviral vector encoding for WAS cDNA under the control of an autologous promoter (1.6 kb). T cells obtained from WAS patients showed normal level of WAS expression after lentiviral transduction. Transduced T cells showed a correction in TCR-driven proliferation and IL-2 production. Furthermore, a selective growth advantage of transduced T cells was observed in long-term in vitro cultures. Studies in T cell clones generated from transduced WAS CD4+ T cells revealed that 1–2 vector copies were necessary and sufficient to correct T cells function. CD34+ cells, isolated from mobilized peripheral blood and bone marrow of healthy donors, were transduced using WASP or GFP-encoding lentiviral vectors. Cells were cultured in the presence of different cytokines to investigate if WAS gene transfer could have any effect on short and long-term differentiation (CFU-C, LTC-IC and B/NK assays). Transduction resulted in a comparable number of CFU-C and LTC-IC colonies and normal B and NK cells differentiation with respect to untransduced cells. Furthermore, transduction of CD34+ cells isolated from the bone marrow of a WAS patient was performed under optimized culture conditions. Lentiviral gene transfer led to restoration of WASP expression in differentiated cells with copy number ranging from 1 to 5 copies per cell. In conclusion, our data demonstrate that the WAS promoter/cDNA-containing lentiviral vector can efficiently transduce and restore WASP expression in CD34+ cells and T cells from WAS patients. Experiments in the Rag2−/−/γchain- murine model are ongoing to test the efficacy and safety of the WASP transduced CD34+ cells. Together, our studies provide a preclinical basis for the implementation of a gene therapy trial for WAS patients.

Impaired Integrin-Dependent Function in Wiskott-Aldrich Syndrome Protein-Deficient Murine and Human Neutrophils

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency that manifests as increased susceptibility to many pathogens. Although the spectrum of infections suffered by WAS patients is consistent with defects in neutrophil (PMN) function, the consequences of WAS protein (WASp) deficiency on this innate immune cell have been unclear. We report that deficiency of WASp in both human and murine PMNs resulted in profound defects in clustering of beta2 integrins, leading to defective adhesion and transendothelial migration under conditions of physiologic shear flow. Wild-type PMNs redistributed clustered beta2 integrins to the uropod of the cell during active migration, whereas WASp-deficient cells remain unpolarized. The WASp-deficient PMNs also showed reduced integrin-dependent activation of degranulation and respiratory burst. PMNs from a WAS patient manifested similar defects in integrin clustering and signaling. These results suggest that impaired beta2 integrin function in WASp-deficient PMNs may contribute substantially to the clinical immunodeficiency suffered by WAS patients.

A second-site mutation in the initiation codon ofWAS (WASP) results in expansion of subsets of lymphocytes in an Wiskott-Aldrich syndrome patient

Wiskott-Aldrich syndrome (WAS) is caused by mutations in the gene encoding WAS protein (WASP ). Recently, somatic mosaicism caused by reversions or second-site mutations has been reported in some inherited disorders including WAS. In this article, we describe somatic mosaicism in a 15-year-old WAS patient due to a second-hit mutation in the initiation codon. The patient originally had a single-base deletion (c.11delG; p.G4fsX40) in the WAS (WASP) gene, which resulted in a frameshift and abrogated protein expression. Subsequently, a fraction of T and natural killer (NK) cells expressed a smaller WASP, which binds to its cellular partner WASP-interacting protein (WIP). The T and NK cells were found to have an additional mutation in the initiation codon (c.1A>T; p.M1_P5del). The results strongly suggest that the smaller WASP is translated from the second ATG downstream of the original mutation, and not only T cells but also NK cells carrying the second mutation acquired a growth advantage over WASP negative counterparts. To our knowledge, this is the first report describing somatic mosaicism due to a second-site mutation in the initiation codon of any inherited disorders.

A novel Wiskott-Aldrich syndrome protein (WASP) complex mutation identified in a WAS patient results in an aberrant product at the C-terminus from two transcripts with unusual polyA signals

Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by immunodeficiency, thrombocytopenia and eczema. A broad spectrum of mutations in the WASP gene has been identified as causing the disease. In the present paper, we report on a patient affected by WAS with a novel complex mutation, characterized by a small 9 bp deletion followed by an inversion of 151 bp and a gross deletion of 4.3 kb within the Xp11.23 region. The small deletion and the inverted fragment are found in intron 11. The large deletion initiates downstream of exon 11 of the WASP gene, including exon 12, and a genomic region upstream of the promoter of the contiguous SUV39H1 gene. Expression studies of the mRNA of the patient's sample showed the presence of two aberrant transcripts that code for a protein of 519 amino acids. We demonstrate that these two transcripts differ in the 3' UTR region, and result from the use of two alternative polyadenylation signals. The severe phenotype of the patient correlates with the presence of an aberrant protein.

WIP-Mediated Mechanisms of Recruitment, Activation and Degradation of WASP in T Cells.

WASP, the product of the gene mutated in Wiskott-Aldrich syndrome (WAS), plays a critical role in T cell activation and actin reorganization. WIP is a WASP-interacting protein that binds to EVH1 domain of WASP, and negatively regulates WASP functions. WASP-mediated actin polymerization and cytoskeletal remodeling are critical for immunological synapse (IS) formation between T cells and antigen presenting cells. To clarify the functions of WIP in T cells, we investigated WIP-mediated mechanisms of recruitment, activation and degradation of WASP following T cell receptor (TCR) ligation and IS formation. WASP translocates to lipid rafts following TCR ligation and localizes at the IS between T cells and B cells presenting superantigen. WASP recruitment is regulated by several TCR signaling pathways mediated by ZAP70-CrkL-WIP and ZAP70-SLP76-Nck complexes. After the recruitment of WASP, WIP is serine-phosphorylated by PKCtheta localized at the IS, and phosphorylated WIP dissociates from WASP, releasing WASP from WIP inhibition. A fraction of WASP is fragmented by Ca-dependent protease calpain and ubiquitinated by Cbl-b, then degraded by the proteasome following TCR ligation. Inhibition of PKCtheta-mediated dissociation of the WASP-WIP complex inhibits WASP degradation. Inhibition of proteasomal degradation results in the accumulation of WASP fragments and enhanced TCR-induced actin polymerization in normal T cells. We demonstrate that WASP protein, but not mRNA, levels are severely diminished in T cells from WIP-deficient mice, but are restored by calpain and proteasome inhibitors. These inhibitors restore WASP protein levels and correct the defect in actin polymerization in response to TCR ligation in T cells from a WAS patient with a missense mutation that disrupts WIP binding. The similarity of the functional defects in WASP- and WIP-deficient mice and the observation that most missense mutations in WAS patients are in the WIP binding EVH1 domain of WASP suggest that WIP may function as a chaperone that controls WASP levels in T cells. These results suggest that WIP is involved in the recruitment and activation of WASP and that degradation of WASP freed from WIP may down-regulate actin polymerization following TCR ligation. More importantly, WIP regulates WASP protein stability in T cells from WAS patients with WASP missense mutations in WIP-binding site and impaired WASP-WIP interaction.

Lymphocyte microvilli are dynamic, actin-dependent structures that do not require Wiskott-Aldrich syndrome protein (WASp) for their morphology

AbstractShort microvilli cover the surfaces of circulating mammalian lymphocytes. The surfaces of monocytes and neutrophils are very different, containing ruffles as their predominant structure. In this study, we present the first quantitative characterization of lymphocyte microvilli. From analysis of scanning electron micrographs, we find that median microvillar length and surface density range from 0.3 to 0.4 μm and 2 to 4 microvilli/μm2, respectively, on lymphocytes from a variety of sources. As with similar structures from other cells, lymphocyte microvilli contain parallel bundles of actin filaments. Lymphocyte microvilli rapidly disassemble when exposed to the actin-sequestering molecule, Latrunculin A. This disassembly parallels cellular actin filament depolymerization and is complete within 2 minutes, suggesting that lymphocyte microvilli undergo continuous assembly and disassembly. In contrast to previous reports suggesting lymphocyte microvillar density to be reduced on lymphocytes from Wiskott-Aldrich syndrome (WAS) patient, we find no such deficiency in either mouse or human WAS protein (WASp)–deficient lymphocytes. These results suggest that WASp is either not involved in or is redundant in the rapid dynamics of lymphocyte microvilli.